No abstract

Secreted proteins, which may be involved in the regulation of various biological processes, are the potential targets for diagnosis and treatment of diverse diseases. In this study, to identify the human hepatoma HepG2 cells-derived secreted proteins more extensively, we applied the protein sample preparations using the combinations of denaturation methods and molecular-mass cutoff via ultrafiltration to the two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS) analysis. We were able to identify a total of 86 proteins containing widely known secreted proteins of HepG2 such as alpha-fetoprotein, of which 73 proteins including 27 signal peptide-containing proteins have never been reported to be secreted from HepG2 cells in other proteomic studies. Among the identified signal peptide-containing proteins, ten proteins such as growth differentiation factor 15, osteopontin and stanniocalcin 2 were discovered as new secreted proteins of HepG2 cells. These observations suggest that the combinations of different sample preparation methods and 2D LC-MS/MS analysis are useful for identifying a wider range of low-abundance proteins and that the secreted proteins from HepG2 identified in this study may be useful as liver-specific biomarkers for diagnosis and treatment.

SUMMARYThe identity of low-molecular-weight and minor protein spots, appeared in 2-DE patterns of human plasma, was examined. They were not obvious in the patterns of "Type-I" 2-DE (non-denaturing IEF followed by non-denaturing gel electrophoresis), but clearly detected in the patterns of "Type II" 2-DE (non-denaturing IEF followed by SDS gel electrophoresis) at pI 5.5-7.5 and apparent mass 8-40 kDa 1) . The spots were not obviously detected when the IEF gels were kept at low temperature (around 4°C) during electrophoresis, suggesting that they are the proteolysis products of plasma proteins. The minor spots were more obviously detected when human plasma was subjected to ammonium sulfate (AS) fractionation and the 0-35% saturated AS fraction was dialyzed and subjected to Type-II 2-DE. Then the 116 spots on the 2-DE pattern, detected at pI 5-7.5 and apparent mass 8-60 kDa, were excised and subjected to MALDI-MS measurements and the mass spectra were analyzed using the software of peptide mass fingerprinting (PMF) Mascot and ProFound to assign the proteins. Many of the spots were assigned to contain fibrinogen α chain, especially those at pI 5.5-7.5 and apparent mass 8-40 kDa, suggesting that these spots are its fragments. The distribution of the MS-detected peptide fragments suggested that the molecular-mass heterogeneity might be caused by the cleavage of multiple sites on the α chain. Care must be taken to keep the temperature of IEF gels at around 4°C during electrophoresis, when human plasma proteins are subjected to non-denaturing IEF. The absence of the spots of fibrinogen fragments on Type-II 2-DE gels would validate the intactness of plasma proteins. The advantages of micro gel system for the analysis of intact protein mixtures are suggested.

ABSTRACT. To analyze the functional differences of the insulin receptor substrate (IRS) family, the N-terminal fragments containing the pleckstrin homology (PH) domains and the phosphotyrosine-binding (PTB) domains of IRS (IRS-N) proteins, as well as intact IRS molecules, were expressed in Cos-1 cells, and insulin-induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS-N fragments non-selectively inhibited insulin-induced tyrosine phosphorylation of IRS-1, IRS-2 and IRS-3, among which IRS3-N was most effective. The mutations of IRS-1 disrupting all the phosphoinositide-binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS-1, which was further inhibited by coexpression of all the IRS-N proteins examined. In contrast, the N-terminal PH domain-interacting region (PHIP-N) of PH-interacting protein (PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS-N proteins, but not PHIP-N, suppressed targeting of IRS-1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity-disrupting mutations of IRS-1 significantly impaired but did not completely abrogate the insulin-induced translocation of IRS-1 to the plasma membrane, which was further suppressed by IRS1-N overexpression. These findings suggest that both insulin-induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.